Methods and compositions for inactivating glutamine synthetase gene expression

ABSTRACT

Disclosed herein are methods and compositions for inactivating a glutamine synthetase (GS) gene, using fusion proteins comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/589,884, filed Oct. 29, 2009, which claims the benefit of U.S.Provisional Application No. 61/197,600, filed Oct. 29, 2008, thedisclosures of which are hereby incorporated by reference in theirentireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the fields of genome engineering, cellculture, generation of cell lines and protein production.

BACKGROUND

Glutamine synthetase (GS) is a critical enzyme in the synthesis of theamino acid L-glutamine. See, Meister, A. in Glutamine Metabolism,Enzymology and Regulation (eds. J. Mora & R. Palacios) 1-40 (AcademicPress, N.Y.; 1980). A GS-negative cell line is therefore auxotrophic forL-glutamine. GS is frequently used as a selection marker gene in CHOcell based recombinant protein expression systems (Wurm et al.(2004)Nature Biotechnology 22: 1393-1398), though the absence of aGS-negative CHO line requires the use of the GS inhibitor methioninesulfoximine to achieve selection.

In addition, dihydrofolate reductase (DHFR,5,6,7,8-tetrahydrofolate:NADP+oxidoreductase) is an essential enzyme inboth eukaryotes and prokaryotes and catalyzes the NADPH-dependentreduction of dihydrofolate to tetrahydrofolate, an essential carrier ofone-carbon units in the biosynthesis of thymidylate, purine nucleotides,glycine and methyl compounds.

DHFR-deficient cells have long been used for production of recombinantproteins. DHFR-deficient cells will only grow in medium supplemented bycertain factors involved in folate metabolism or if DHFR is provided tothe cell, for example as a transgene. Cells into which a DHFR transgenehas been stably integrated can be selected for by growing the cells inunsupplemented medium. Moreover, exogenous sequences are typicallyco-integrated when introduced into a cell using a single polynucleotide.Accordingly, when the DHFR transgene also includes a sequence encoding aprotein of interest, selected cells will express both DHFR and theprotein of interest. Furthermore, in response to inhibitors such asmethotrexate (MTX), the DHFR gene copy number can be amplified.Accordingly, sequences encoding a protein of interest that areco-integrated with exogenous DHFR can be amplified by gradually exposingthe cells to increasing concentrations of methotrexate, resulting inoverexpression of the recombinant protein of interest. However, despitethe wide use of DHFR-deficient cell systems for recombinant proteinexpression, currently available DHFR-deficient cell lines do not grow aswell as the parental DHFR-competent cells from which they are derived.

Thus, mammalian cells with single and multi-gene knockouts have enormousutility in research, drug discovery, and cell-based therapeutics.However, conventional methods for the targeted elimination of aninvestigator-specified gene rely upon the process of homologousrecombination or gene targeting. Mansour et al. (1988) Nature336:348-352; Vasquez et al. (2001) Proc Natl Acad Sci USA 98:8403-8410;Rago et al. (2007) Nature Protocols 2:2734-2746; Kohli et al. (2004)Nucleic Acids Research 32, e3. While capable of generating a definedbiallelic knockout, for many cell types this technique has proven tooinefficient and thus too laborious for routine application. See, e.g,Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-622. These methodsfor targeted gene deletion require sequential rounds of homologousrecombination and drug selection to isolate rare desired events—aprocess sufficiently laborious to limit application to individual loci.Consequently, the generation of mammalian cell lines modified atmultiple target loci has been largely unexplored.

Zinc-finger nucleases (ZFNs) have been used for targeted cleavage andgene inactivation. See, for example, United States Patent Publications20030232410; 20050208489; 20050026157; 20050064474; 20060188987;20060063231; 2008/0015164 and U.S. Ser. No. 12/218,035 and InternationalPublication WO 07/014,275, the disclosures of which are incorporated byreference in their entireties for all purposes. Formed via the fusion ofan engineered zinc-finger DNA binding domain specific for a designatedtarget sequence and the catalytic domain of Fok I (a restrictionendonuclease from Flavobacterium okeanokoites), ZFNs provide the abilityto place a double-strand DNA break (DSB) at a chosen genomic address.The removal of this site-specific DSB is carried out by the cell's ownDNA repair machinery either via a homology-directed repair process whendonor DNA is provided, or via non-homologous end joining (NHEJ). See,e.g., Urnov et al. (2005)Nature 435:646-651 (2005); Moehle et al. (2007)Proc Natl Acad Sci USA 104:3055-3060 (2007); Bibikova, et al. (2001) MolCell Biol 21:289-297; Bibikova et al. (2003) Science 300:764; Porteus etal. (2005) Nature Biotechnology 23:967-973; Lombardo et al. (2007)Nature Biotechnology 25:1298-1306; Perez et al. (2008) NatureBiotechnology 26:808-816; Bibikova et al. (2002) Genetics 161:1169-1175;Lloyd et al. (2005) Proc Natl Acad Sci USA 102:2232-2237; Morton et al.(2006) Proc Natl Acad Sci USA 103:16370-16375.

While both homology-directed repair and NHEJ processes result in themodification of the target locus, the NHEJ-driven approach obviates theneed for donor DNA design and synthesis yet results in a high frequencyof disrupted alleles and the error-prone nature of the NHEJ-mediated DSBrepair can be exploited to achieve the knockout of a targeted gene inmammalian cells following simple transient transfection of a DNAconstruct encoding the ZFNs. See, e.g., Santiago et al. (2008) Proc NatlAcad Sci USA 105:5809-5814. ZFN technology has allowed the isolation ofseveral independent knockout cell lines from a screening effort of lessthan one 96-well plate of single-cell derived clones. As no donor DNA orselection strategy was employed, the resultant single-gene knock outline is a suitable starting cell line for subsequent geneticmodification.

SUMMARY

Disclosed herein are compositions for the partial or completeinactivation of a GS gene. Also disclosed herein are methods of makingand using these compositions (reagents), for example to inactivate GS ina cell to produce cell lines in which a GS gene is inactivated. GSdisrupted cell lines are useful, for example, in production ofrecombinant proteins.

In one aspect, zinc finger proteins, engineered to bind in a GS gene,are provided. Any of the zinc finger proteins described herein mayinclude 1, 2, 3, 4, 5, 6 or more zinc fingers, each zinc finger having arecognition helix that binds to a target subsite in a GS gene. Incertain embodiments, the zinc finger proteins comprise 4, 5 or 6 fingers(wherein the individual zinc fingers are designated F1, F2, F3, F4, F5and F6) and comprise the amino acid sequence of the recognition helicesshown in Table 1.

In another aspect, zinc finger proteins, engineered to bind in a DHFRgene, are provided. Any of the zinc finger proteins described herein mayinclude 1, 2, 3, 4, 5, 6 or more zinc fingers, each zinc finger having arecognition helix that binds to a target subsite in a DHFR gene. Incertain embodiments, the zinc finger proteins comprise 4, 5 or 6 fingers(wherein the individual zinc fingers are designated F1, F2, F3, F4, F5and F6) and comprise the amino acid sequence of the recognition helicesshown in Table 2.

In another aspect, fusion proteins comprising any of the zinc fingerproteins described herein and at least one cleavage domain or at leastone cleavage half-domain, are also provided. In certain embodiments, thecleavage half-domain is a wild-type Fokl cleavage half-domain. In otherembodiments, the cleavage half-domain is an engineered Fold cleavagehalf-domain.

In yet another aspect, a polynucleotide encoding any of the proteinsdescribed herein is provided.

In yet another aspect, also provided is an isolated cell comprising anyof the proteins and/or polynucleotides as described herein. In certainembodiments, GS is inactivated (partially or fully) in the cell. Any ofthe cells described herein may include additional genes that have beeninactivated, for example, using zinc finger nucleases designed to bindto a target site in the selected gene. In certain embodiments, providedherein are cells or cell lines in which FUT8, dihydrofolate reductase(DHFR) and glutamine synthetase (GS) have been inactivated.

In addition, methods of using the zinc finger proteins and fusionsthereof in methods of inactivating GS in a cell or cell line areprovided.

Thus, in another aspect, provided herein is a method for inactivating acellular GS gene (e.g., an endogenous GS gene) in a cell, the methodcomprising: (a) introducing, into a cell, a first nucleic acid encodinga first polypeptide, wherein the first polypeptide comprises: (i) a zincfinger DNA-binding domain that is engineered to bind to a first targetsite in an endogenous GS gene; and (ii) a cleavage domain; such that thepolypeptide is expressed in the cell, whereby the polypeptide binds tothe target site and cleaves the GS gene. In certain embodiments, themethods further comprise introducing a nucleic acid encoding a secondpolypeptide, wherein the second polypeptide comprises: (i) a zinc fingerDNA-binding domain that is engineered to bind to a second target site inthe GS gene; and (ii) a cleavage domain; such that the secondpolypeptide is expressed in the cell, whereby the first and secondpolypeptides bind to their respective target sites and cleave the GSgene. The first and second polypeptides may be encoded by the firstnucleic acid or by different nucleic acids. In certain embodiments, oneor more additional polynucleotides or polypeptides are introduced intothe cells, for example polynucleotides encoding additional zinc fingerproteins.

In yet another aspect, the disclosure provides a method of producing arecombinant protein of interest in a host cell, the method comprisingthe steps of: (a) providing a host cell comprising an endogenous GSgene; (b) inactivating the endogenous GS gene of the host cell by any ofthe methods described herein; and (c) introducing an expression vectorcomprising a transgene, the transgene comprising a sequence encoding aprotein of interest, into the host cell, thereby producing therecombinant protein. In certain embodiments, the protein of interestcomprises an antibody, e.g., a monoclonal antibody.

In any of the cells and methods described herein, the cell or cell linecan be for example, but not limited to a COS, CHO (e.g., CHO—S, CHO-K1,CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79,B14AF28-G3, BHK, HaK, NSO, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F,HEK293-H, HEK293-T), NIH3T3, perC6, insect cell such as Spodopterafugzperda (Sf), or fungal cell such as Saccharomyces, Pichia andSchizosaccharomyces.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, panels A to E, show ZFN-mediated disruption of the glutaminesynthetase gene in CHO cells and generation of single knockout GS−/−cell lines. FIG. 1A is a schematic depicting the actively-transcribed GSgene, which contains 7 exons. The start codon ATG located in exon 2.FIG. 1A also shows that the target sequence for ZFN pair ZFN9372/ZFN9075is located in exon 6 as indicated and that the target sequence of ZFNpair 8361/8365 is located in exon 2. Additional ZFNs (9076, 9179, 7858,7889, 9373) targeted to exon 6 are also shown. FIG. 1B are gels showingZFN-mediated disruption of the endogenous CHO GS gene using ZFN pair9372/9075 linked to either the wild-type or an obligate heterodimerEL/KK variant of the catalytic domain of Fok I in CHO-K¹ cells (leftpanel) or CHO—S cells (right panel), as determined by the Surveyor™Nuclease Assay. FIG. 1C (SEQ ID NOS: 58-73, respectively) depictsexemplary DNA sequences of the target GS locus for the indicated celllines along with their growth properties in the absence of exogenouslyadded L-glutamine (“L-Glu Growth”; ‘+’: growth in the absence ofL-glutamine indistinguishable from wild-type CHO; ‘−’: no growth in theabsence of L-glutamine). The ZFN target sequences are underlined. Theprotein translation is shown under the wild-type sequence. Capitalletters indicate exonic sequences, small letters indicate intronicsequences, ‘−’ indicate deletions, bold letters indicate insertions.FIG. 1D depicts Western blot analysis of selected CHO—S (left panel) andCHO-K1 (right panel) cell lines using an anti-GS monoclonal antibody(top panels). As a loading control, blots were re-probed with ananti-DHFR antibody (bottom panels). FIG. 1E shows graphs depictinggrowth and viability of selected CHO-K1 GS−/− cell lines that had beengrown for period of approximately 3 months. The viability (smooth lineon the top of the graph) and viable cell density (jagged line) of thesecell lines is shown for a 30 day period following the initial 3 monthperiod, where cells were grown in L-glutamine-supplemented medium,splitting the cells every 2-3 days. L-glutamine was withdrawn at thetime indicated by the arrow.

FIG. 2, panels A to F, depict ZFN-mediated disruption of the DHFR genein CHO cells and generation of double knockout DHFR^(−/−)GS^(−/−) celllines. FIG. 2A shows the genomic organization of the DHFR gene in CHOcells and the location of the target sites for ZFN pairs 9461/7844 (inexon 1, see, U.S. Patent Application No. 20080015164) and 9476/9477 (inintron 1 located 240-bp 3′ of the exon 1 ZFN 9461/7844 cleavage site).FIG. 2B depicts the level of gene modification using ZFN pair 9461/7844(left panel) and ZFN pair 9476/9477 (right panel), as measured using theSurveyor™ Nuclease Assay. FIG. 2 C depicts PCR analysis of GS^(−/−)clone B3 genomic DNA 2 days after concurrent transfection of plasmidsencoding both ZFN pairs, which resulted in a deletion of approximately240 by in the DHFR locus (small arrow in lane 7). Serving as controlswere cells transfected with empty vector (Lane 1), a GFP control vector(Lane 2), the exonic ZFN pair only (ZFN9461/ZFN7844, Lane 3), theintronic ZFN pair only (ZFN9476/ZFN9477, Lane 4), the “inner” ZFNsrelative to the deletion (ZFN7844 and ZFN9477, Lane5), and the “outer”ZFNs relative to the deletion (ZFN9461 and ZFN9476, Lane 6). FIG. 2D(SEQ ID NOS: 74-81, respectively) depicts results of DHFR genotypinganalysis of single-cell lines. For homozygous clones, the commonsequence for both alleles is shown. For compound heterozygous clones thesequence of each unique allele is shown. FIG. 2E depicts Western blotanalysis with the indicated antibodies (DHFR, GS and (β-tubulin) ofwhole cell lysates from the cell lines indicated above each lane. Celllines 1F1.6 and 2B12.8 (DHFR/GS knockout) were analyzed while theparental GS^(−/−) cell line B3, wild-type CHO cells (WT), and theDHFR-deficient CHO cell line DG44 (see Urlab et al. (1983) Cell33:405-412) served as controls. FIG. 2F depicts growth of the 1F1.6 and2B12.8 cell lines and the dependence of these lines on exogenouslyprovided hypoxanthine, thymidine and glutamine.

FIG. 3, panels A to D, show ZFN-mediated disruption of the FUT8 gene inCHO cells and generation of triple knockout FUT8^(−/−) DHFR^(−/−)Gg^(−/−) cell lines. FIG. 3A is a schematic depicting ZFNs targeted tothe critical and highly conserved region encoding the FUT8 Fut motif IIlocated in exon 10. See, also, U.S. patent application Ser. No.12/218,0135. FIG. 3B depicts the level of gene modification 2 days aftertransient transfection of the ZFN pair 12176/12172 into the DHFR^(−/−)GS^(−/−) clone 1F1.6, as measured using the Surveyor™ Nuclease Assay.FIG. 3C depicts fluorescent-LCA binding activity of the triple-KO clones35F2 and 14C1 (lines on left side of panel) by FACS analysis. F-LCAstained wild-type CHO—S cells (line on right side of panel) served aspositive controls, and unstained cells (dotted line) served as negativecontrols. FIG. 3D (SEQ ID NOS: 82-87, respectively) shows the genotype(for both alleles) of the triple-knockout cell lines 35F2 and 14C1 atthe FUT8 locus where the sequence shown is for both alleles. Capitalletters indicate exon 10 sequences, small letters indicates intronsequences, italic letters indicate Fut motif II sequences, bold lettersindicate sequence insertions, ‘−’ indicates deletions. The proteintranslation is also shown under the wild-type sequence. Underlineindicates the ZFN binding sites.

FIG. 4, panels A to C, depict an exemplary ZFN design and its targetsite in the CHO GS gene. FIG. 4A is a schematic representation of thefunctional intron-containing glutamine synthetase gene in CHO cells. Itcontains 7 exons, the start codon ATG is located in exon 2, and thetarget sequence of ZFN pair ZFN9372/ZFN9075, located in exon 6, is alsoindicated. The nucleotide sequence (SEQ ID NO: 88) of the region ofinterest around exon 6 is shown. Capital letters indicate exon 6sequences, small letters indicate intron sequences. The target sequencesof ZFN9372 and ZFN9075 are underlined. FIG. 4B (SEQ ID NOS: 89 and 90)is a schematic representation of ZFN9372 and ZFN9075 binding to theirdouble-stranded target sequence (underlined). FIG. 4C depicts the targetsequences and zinc-finger designs of ZFN9372 (SEQ ID NOS: 6-12) andZFN9075 (SEQ ID NOS: 1-5). The core DNA target sequences (capitalletters) and 2 flanking bases (small letters) are shown. ZFN9075contains 4 zinc-finger DNA-binding domains, and ZFN9372 contains 6zinc-finger domains. The amino acid residues at positions ‘−1’ to ‘+F6’of the recognition a-helix of each of the zinc-finger DNA-bindingdomains for the indicated target DNA triplets are shown.

FIG. 5 (SEQ ID NOS: 91-132, respectively) shows exemplary results ofsequencing of the genomic GS locus from ZFN transfected cells. “C”refers to the count (number of times the indicated sequence wasobserved) and “G” refers to the genotype. The ZFN target sequences areunderlined. Bold letters indicate sequence insertions, ‘−’ indicatesdeletions.

FIG. 6 is a graph depicting L-glutamine-dependent growth of a homozygousGS^(−/−) cell line (Clone B3). In the presence of L-glutamine, CHO—SGS^(−/−) clone B3 (triangle) grows as well as wild-type CHO—S (diamond).In the absence of exogenous L-glutamine, the Clone B3 (circle) stoppedgrowing and all cells died within 4 days, whereas the wild-type CHOcells (square) continued to grow at a reduced rate.

FIG. 7, panels A to C, depict exemplary ZFN designs and their targetsites in the CHO DHFR gene. FIG. 7A (SEQ ID NO: 133) shows thenucleotide sequence of the DHFR region targeted by the indicated ZFNpairs in CHO cells. Capital letters indicate exon sequences, smallletters indicate intron sequences. FIG. 7B (SEQ ID NOS: 134-137,respectively) is a schematic representation of the ZFNs targeting DHFRwhen the ZFNs are bound to their double-stranded target sequences(underlined). The two ZFNs labeled “inner” target sequences are internalto the region to be deleted, while the two ZFNs labeled “outer” targetsequences are outside of the expected deletion junction. FIG. 7C showsthe target sequences (SEQ ID NOS: 33, 32, 38, and 41, respectively) andzinc-finger designs (SEQ ID NOS: 37, 36, 35, 34, 25, 2, 3, 14, 40, 14,39, 2, 43, 42, 17, and 7, respectively) of exemplary ZFNs targeting theCHO DHFR gene. The core DNA target sequences (capital letters) and 2flanking bases (small letters) are shown. All ZFNs contain 4 zinc-fingerDNA-binding domains. The amino acid residues at positions ‘−1’ to ‘+F6’of the recognition a-helix for each of the zinc-finger DNA-bindingdomains for the indicated target DNA triplet are shown.

FIG. 8 (SEQ ID NOS: 138-150, respectively) shows exemplary results ofsequencing of the genomic DHFR locus from ZFN transfected cells. “C”refers to the count (number of times the indicated sequence wasobserved) and “G” refers to the genotype. The ZFN target sequences areunderlined. Bold letters indicate sequence insertions, ‘−’ indicatesdeletions. Bold letters indicate sequence insertions. Italics indicatesequence changes. Capital letters indicate exonic sequence, smallletters indicate intronic sequence.

FIG. 9, panels A to C, depict ZFN target sites and finger designstargeted to the CHO FUT8 gene. FIG. 9A (SEQ ID NO: 151) depicts aschematic of the ZFN binding sites within exon 10 of the FUT8 gene aswell as the nucleotide sequence of the target region within exon 10.Capital letters indicate exon 10 sequences, small letters indicateintronic sequences. The target sequences of ZFN12176 and ZFN12172 areunderlined. The nucleotides comprising the fucosyltransferase motif IIare shown in italics. FIG. 9B is a schematic representation of ZFN12176(SEQ ID NO: 153) and ZFN12172 (SEQ ID NO: 152) binding to theirdouble-stranded target sequence (underlined). FIG. 9C depicts the targetsequences (SEQ ID NOS: 154 and 155) and zinc-finger designs (SEQ ID NOS:54, 53, 52, 51, 3, 49, 48, 47, 9, 46, and 45, respectively)⁻of ZFN12176and ZFN12172. The core DNA target sequences (capital letters) and 2flanking bases (small letters) are shown. ZFN12176 contains 5zinc-finger DNA-binding domains, and ZFN12172 contains 6 zinc-fingerdomains. The amino acid residues at positions ‘−1’ to ‘+6’ of therecognition α-helix for each of the zinc-finger DNA-binding domains forthe indicated target DNA triplet are shown.

FIG. 10, panels A to C, show ZFN targeting of CCR5, GR and AAVS1 loci.FIG. 10A is a schematic showing the location of ZFN targeting sites inC—C chemokine receptor 5 (CCR5), glucocorticoid receptor (GR), andadeno-associated virus integration site (AAVS1) loci, respectively. FIG.10B depicts ZFN-mediated simultaneous disruption, as measured bySurveyor™ Nuclease Assay, of CCR5 (left panel), GR (middle panel), andAAVS1 (right panel) loci as measured 20 days after transientco-transfection of pairs of ZFNs linked to either the wild-type, ZFN-FokI (wt) or an obligate heterodimer EL/KK variant, ZFN-Fok I(EL/KK), ofthe catalytic domain of Fok into K562 cells. Treatments for each laneare as following: lanel, CCR5ZFN-FokI (wt); lane 2, GR ZFN-Fok I(wt);lane 3, AA VS1 ZFN-Fok I(wt); lane 4, CCR5ZFN-Fok I(EL/KK); lane 5, GRZFN-Fok I(EL/KK); lane 6, AA VS1 ZFN-Fok I(EL/KK); lane 7,CCR5ZFN-FokI(wt)+GR ZFN-FokI(wt)+AAVS1 ZFN-Fok/(wt); lane 8, CCR5ZFN-FokI(EL/KK)+GR ZFN-Fok/(EL/KK)+AAVS1 ZFN-Fok/(EL/KK). FIG. 10C (SEQID NOS: 156-165, respectively) depicts the genotype of a single tripleknockout clone wherein the target CCR5, GR, AAVS1 loci for the indicatedcell lines are shown. The ZFN target sequences are underlined. ‘−’indicate deletions, bold letters indicate insertions.

FIG. 11, panels A and B, show sequence alignments of CHO GS genomicsequences as determined herein with the indicated species. FIG. 11A (SEQID NOS: 166-174, respectively) shows alignment of exon 2 sequence. Thetarget sites for ZFN8361 and ZFN8365 are underlined. FIG. 11B (SEQ IDNOS: 175-185, respectively) shows alignment of exon 6 sequence. Thetarget sites for ZFN9075 and ZFN9372 are underlined.

FIG. 12, panels A to D, show ZFN-mediated disruption in human and mousecells. FIGS. 12A and B show disruption of GS in human K562 mediated byZFNs targeted to exon 2 of CHO GS (FIG. 12A) and exon 6 of CHO GS (FIG.12B). FIGS. 12C and D show disruption of GS in mouse Neuro2a cellsmediated by ZFNs targeted to exon 2 of CHO GS (FIG. 12C) and exon 6 ofCHO GS (FIG. 12D).

FIG. 13, panels A to C, show the complete genomic sequence of CHO GSlocus (SEQ ID NO:55). Introns and exons are designated as shown.

FIG. 14, panels A to C, show the results of GS-specific gene targetingin HEK293 cells. FIG. 14A depicts the percent of NHEJ activity in cellstreated with GS-specific ZFNs (labeled GS) in comparison with cellstransfected with a GFP donor molecule (labeled GFP). FIG. 14B shows that2 clones, g17 and g52, derived from a pool of cells treated with theGS-specific ZFNs, do not express GS as assayed by Western blot.

FIG. 14C is a graph showing growth over 7 days of the GS knock outclones and demonstrates the requirement for glutamine supplementationfor growth.

DETAILED DESCRIPTION

Described herein are compositions and methods for partial or completeinactivation of a GS gene. Also disclosed are methods of making andusing these compositions (reagents), for example to inactivate a GS genein a target cell. Inactivation of GS, alone or in combination with othergenes such as DHFR and FUT8 in a target cell can be used to produce celllines for expression of recombinant proteins, for example monoclonalantibodies that elicit enhanced antibody-dependent cellular cytotoxicity(ADCC).

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P.M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P.B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence. Non-limiting examples of methods forengineering zinc finger proteins are design and selection. A designedzinc finger protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP designs and binding data. See, for example,U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection. See e.g., U.S. Pat. No.5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat.No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197 and WO02/099084.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. An approximatealignment for nucleic acid sequences is provided by the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981). This algorithm can be applied to amino acid sequencesby using the scoring matrix developed by Dayhoff, Atlas of ProteinSequences and Structure, M.O. Dayhoff ed., 5 suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplaryimplementation of this algorithm to determine percent identity of asequence is provided by the Genetics Computer Group (Madison, Wis.) inthe “BestFit” utility application. Other suitable programs forcalculating the percent identity or similarity between sequences aregenerally known in the art, for example, another alignment program isBLAST, used with default parameters. For example, BLASTN and BLASTP canbe used using the following default parameters: genetic code=standard;filter=none; strand=both; cutoff =60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found on theGenBank website. With respect to sequences described herein, the rangeof desired degrees of sequence identity is approximately 80% to 100% andany integer value therebetween. Typically the percent identities betweensequences are at least 70-75%, preferably 80-82%, more preferably85-90%, even more preferably 92%, still more preferably 95%, and mostpreferably 98% sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system.

Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: APractical Approach, editors B.D. Hames and S.J. Higgins, (1985) Oxford;Washington, D.C.; IRL Press).

Selective hybridization of two nucleic acid fragments can be determinedas follows. The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit the hybridization of a completely identicalsequence to a target molecule. Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern (DNA) blot, Northern(RNA) blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a reference nucleic acidsequence, and then by selection of appropriate conditions the probe andthe reference sequence selectively hybridize, or bind, to each other toform a duplex molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a reference sequence under moderatelystringent hybridization conditions typically hybridizes under conditionsthat allow detection of a target nucleic acid sequence of at least about10-14 nucleotides in length having at least approximately 70% sequenceidentity with the sequence of the selected nucleic acid probe. Stringenthybridization conditions typically allow detection of target nucleicacid sequences of at least about 10-14 nucleotides in length having asequence identity of greater than about 90-95% with the sequence of theselected nucleic acid probe. Hybridization conditions useful forprobe/reference sequence hybridization, where the probe and referencesequence have a specific degree of sequence identity, can be determinedas is known in the art (see, for example, Nucleic Acid Hybridization: APractical Approach, editors B. D. Hames and S. J. Higgins, (1985)Oxford; Washington, D.C.; IRL Press).

Conditions for hybridization are well-known to those of skill in theart. Hybridization stringency refers to the degree to whichhybridization conditions disfavor the formation of hybrids containingmismatched nucleotides, with higher stringency correlated with a lowertolerance for mismatched hybrids. Factors that affect the stringency ofhybridization are well-known to those of skill in the art and include,but are not limited to, temperature, pH, ionic strength, andconcentration of organic solvents such as, for example, formamide anddimethylsulfoxide. As is known to those of skill in the art,hybridization stringency is increased by higher temperatures, lowerionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of the sequences, base composition of thevarious sequences, concentrations of salts and other hybridizationsolution components, the presence or absence of blocking agents in thehybridization solutions (e.g., dextran sulfate, and polyethyleneglycol), hybridization reaction temperature and time parameters, as wellas, varying wash conditions. The selection of a particular set ofhybridization conditions is selected following standard methods in theart (see, for example, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.).

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

An “cleavage half-domain” is a polypeptide sequence which, inconjunction with a second polypeptide (either identical or different)forms a complex having cleavage activity (preferably double-strandcleavage activity). The terms “first and second cleavage half-domains;”“+ and − cleavage half-domains” and “right and left cleavagehalf-domains” are used interchangeably to refer to pairs of cleavagehalf-domains that dimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474; 2007/0218528 and2008/0131962, incorporated herein by reference in their entireties.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H₂B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases. An exogeneous molecule can also be the same type of moleculeas an endogenous molecule but derived from a different species than thecell is derived from. For example, a human nucleic acid sequenced may beintroduced into a cell line originally derived from a mouse or hamster.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule, can also refer to a nucleic acid from a differentspecies, for example, a human gene inserted into a hamster genome.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, shRNAs, micro RNAs (miRNAs) ribozyme, structural RNA or any othertype of RNA) or a protein produced by translation of a mRNA. Geneproducts also include RNAs which are modified, by processes such ascapping, polyadenylation, methylation, and editing, and proteinsmodified by, for example, methylation, acetylation, phosphorylation,ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Gene inactivation refers to anyreduction in gene expression as compared to a cell that does not includea ZFP as described herein. Thus, gene inactivation may be complete(knock-out) or partial (e.g., a hypomorph in which a gene exhibits lessthan normal expression levels or a product of a mutant gene that showspartial reduction in the activity it influences).

“Eucaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to a cleavage domain, the ZFP DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

The term “antibody” as used herein includes antibodies obtained fromboth polyclonal and monoclonal preparations, as well as, the following:hybrid (chimeric) antibody molecules (see, for example, Winter et al.,Nature (1991) 349:293-299; and U.S. Pat. No. 4,816,567); F(ab′)₂ andF(ab) fragments; Fv molecules (non-covalent heterodimers, see, forexample, Inbar et al., Proc Natl Acad Sci USA (1972) 69:2659-2662; andEhrlich et al., Biochem (1980) 19:4091-4096); single-chain Fv molecules(sFv) (see, for example, Huston et al., Proc Natl Acad Sci USA (1988)85:5879-5883); dimeric and trimeric antibody fragment constructs;minibodies (see, e.g., Pack et al., Biochem (1992) 31:1579-1584; Cumberet al., J Immunology (1992) 149B: 120-126); humanized antibody molecules(see, for example, Riechmann et al., Nature (1988) 332:323-327;Verhoeyan et al., Science (1988) 239:1534-1536; and U.K. PatentPublication No. GB 2,276,169, published 21 Sep. 1994); and, anyfunctional fragments obtained from such molecules, wherein suchfragments retain immunological binding properties of the parent antibodymolecule.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. The termencompasses whole immunoglobulins as well as fragments such as Fab,F(ab′)₂, Fv, and other fragments, as well as chimeric and humanizedhomogeneous antibody populations that exhibit immunological bindingproperties of the parent monoclonal antibody molecule.

Zinc Finger Nucleases

Described herein are zinc finger nucleases (ZFNs) that can be used forinactivation of a GS gene. ZFNs comprise a zinc finger protein (ZFP) anda nuclease (cleavage) domain.

A. Zinc Finger Proteins

Zinc finger binding domains can be engineered to bind to a sequence ofchoice. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan etal. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr.Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct.Biol. 10:411-416. An engineered zinc finger binding domain can have anovel binding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, for example, co-owned U.S. Pat. Nos.6,453,242 and 6,534,261, incorporated by reference herein in theirentireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. PatentApplication Publication Nos. 20050064474 and 20060188987, incorporatedby reference in their entireties herein.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein. Examples of additional linker structures are found inU.S. Provisional Application No. 61/130,099, filed May 28, 2008 andentitled Compositions For Linking DNA-Binding Domains And CleavageDomains.

Table 1 describes a number of zinc finger binding domains that have beenengineered to bind to nucleotide sequences in the GS gene. See, also,FIG. 1 and FIG. 3. Each row describes a separate zinc finger DNA-bindingdomain. The DNA target sequence for each domain is shown in the firstcolumn and the second through fifth columns show the amino acid sequenceof the recognition region (amino acids -1 through +6, with respect tothe start of the helix) of each of the zinc fingers (F1 through F4, F5or F6) in the protein. Nucleotides shown in uppercase denote tripletsthat are directly targeted by a zinc finger and nucleotides shown inlower case denotes bases that are not directly targeted by a zinc finger(for example due to longer linkers between fingers which can result in“skipping” a base). Also provided in the first column is anidentification number for the proteins.

TABLE 1 Zinc finger nucleases targeted to GS ZFN name Target sequence F1F2 F3 F4 F5 F6 ZFN 9075 QSSDLSR RSDNLRE RSDTLSN RKDVRIT gaATGGTGCAGGCTgc(SEQ ID (SEQ ID (SEQ ID (SEQ ID N/A N/A (SEQ ID NO: 1) NO: 2) NO: 3)NO: 4) NO: 5) ZFN 9372 RSDHLST QSSDLRR RSDHLSQ QSANRTT RSDNLSQ ASNDRKKgtTCCCAGGAATGGG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID CTTGGggNO: 7) NO: 8) NO: 9) NO: 10) NO: 11) NO: 12) (SEQ ID NO: 6) ZFN 8361QSGALAR RSDALTQ RSDSLSA RSAHLSR N/A N/A ctGGGTTGATGGTActg (SEQ ID(SEQ ID (SEQ ID (SEQ ID gagaaggactg NO: 14) NO: 15) NO: 16) NO: 17)(SEQ ID NO: 13) ZFN 8365 RSDHLST QSSDLRR RSDSLSV DNANRTK N/A N/AtaTACATGGCTTGGact (SEQ ID (SEQ ID (SEQ ID (SEQ ID ttctcaccctg NO: 7)NO: 8) NO: 19) NO: 20) (SEQ ID NO: 18) ZFN 9076 QSSDLSR RSDNLRE RSSALTRRSDALTQ N/A N/A gaATGGTGCAGGCTgc (SEQ ID (SEQ ID (SEQ ID (SEQ IDcataccaacttt NO: 2) NO: 3) NO: 22) NO: 15) (SEQ ID NO: 21) ZFN 9179QSSDLSR RSDNLRE QSSHLTR TSSNRKT N/A N/A ggAATGGTgCAGGCTg (SEQ ID (SEQ ID(SEQ ID (SEQ ID ccataccaactt NO: 2) NO: 3) NO: 24) NO: 25)(SEQ ID NO: 23) ZFN 7858 QSSDLSR RSDNLRE RSDSLLR RSDALTQ N/A N/AgaATGGTGCAGGCTgc (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 1) NO: 2)NO: 3) NO: 26) NO: 15) ZFN 7889 RSDHLSQ QSANRTT RSDNLSQ ASNDRKK N/A N/AgtTCCCAGGAATGGgc (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 27) NO: 9)NO: 10) NO: 11) NO: 12) ZFN 9373 RSDHLSQ RNADRIT RSDHLSQ QSANRTT RSDNLSQASNDRKK gtTCCCAGGAATGGgC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDTTGGGgtcaaag NO: 9) NO: 29) NO: 9) NO: 10) NO: 11) NO: 12)(SEQ ID NO: 28)

As described below, in certain embodiments, a four-, five-, orsix-finger binding domain as shown in Table 1 is fused to a cleavagehalf-domain, such as, for example, the cleavage domain of a Type IIsrestriction endonuclease such as FokI. One or more pairs of such zincfinger/nuclease half-domain fusions are used for targeted cleavage, asdisclosed, for example, in U.S. Patent Publication Nos. 20050064474 and20070218528.

For targeted cleavage, the near edges of the binding sites can separatedby 5 or more nucleotide pairs, and each of the fusion proteins can bindto an opposite strand of the DNA target. All pairwise combinations ofthe proteins shown in Table 1 can be used for targeted cleavage of a GSgene. Following the present disclosure, ZFNs can be targeted to anysequence in a GS gene.

B. Cleavage Domains

The ZFNs also comprise a nuclease (cleavage domain, cleavagehalf-domain). The cleavage domain portion of the fusion proteinsdisclosed herein can be obtained from any endonuclease or exonuclease.Exemplary endonucleases from which a cleavage domain can be derivedinclude, but are not limited to, restriction endonucleases and homingendonucleases. See, for example, 2002-2003 Catalogue, New EnglandBiolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., S1Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease;yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, ColdSpring Harbor Laboratory Press, 1993). One or more of these enzymes (orfunctional fragments thereof) can be used as a source of cleavagedomains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a)Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fok Ifusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014,275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003)Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474; 20060188987 and20080131962, the disclosures of all of which are incorporated byreference in their entireties herein. Amino acid residues at positions446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531,534, 537, and 538 of Fok I are all targets for influencing dimerizationof the Fok I cleavage half-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:1499L”. As describedin the examples a pair of ZFNs in which one ZFN comprises the“E490K:1538K” cleavage domain and other comprises “Q486E:1499L” cleavagedomain is also referred to as a “EL/KK” ZFN pair. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished when one or, morepairs of nucleases containing these cleavage half-domains are used forcleavage. See, e.g., U.S. Patent Publication No. 20080131962, thedisclosure of which is incorporated by reference in its entirety for allpurposes.

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for, example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474 (Example 5) and 20070134796 (Example 38).

C. Additional Methods for Targeted Cleavage in GS

Any nuclease having a target site in a GS gene can be used in themethods disclosed herein. For example, homing endonucleases andmeganucleases have very long recognition sequences, some of which arelikely to be present, on a statistical basis, once in a human-sizedgenome. Any such nuclease having a unique target site in a GS gene canbe used instead of, or in addition to, a zinc finger nuclease, fortargeted cleavage in a GS gene.

Exemplary homing endonucleases include I-SceI,I-CeuI, PI-PspI, PI-Sce,I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI,I-TevII and I-TevIII. Their recognition sequences are known. See alsoU.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997)Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118;Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996)Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue.

Although the cleavage specificity of most homing endonucleases is notabsolute with respect to their recognition sites, the sites are ofsufficient length that a single cleavage event per mammalian-sizedgenome can be obtained by expressing a homing endonuclease in a cellcontaining a single copy of its recognition site. It has also beenreported that the specificity of homing endonucleases and meganucleasescan be engineered to bind non-natural target sites. See, for example,Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003)Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66. Thus,engineered homing endonucleases and/or meganucelases which are designedto specifically bind a desired target locus, such as GS, may also beused.

Delivery

The ZFNs described herein may be delivered to a target cell by anysuitable means. Suitable cells include but not limited to eukaryotic andprokaryotic cells and/or cell lines. Non-limiting examples of such cellsor cell lines include COS, CHO (e.g., CHO—S, CHO-K1, CHO-DG44,CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK,HaK, NSO, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T),and perC6 cells as well as insect cells such as Spodoptera fugiperda(Sf), or fungal cells such as Saccharomyces, Pichia andSchizosaccharomyces.

Methods of delivering proteins comprising zinc fingers are described,for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261;6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539;7,013,219; and 7,163,824, the disclosures of all of which areincorporated by reference herein in their entireties.

ZFNs as described herein may also be delivered using vectors containingsequences encoding one or more of the ZFNs. Any vector systems may beused including, but not limited to, plasmid vectors, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc. See, also, U.S. Pat.Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219;and 7,163,824, incorporated by reference herein in their entireties.Furthermore, it will be apparent that any of these vectors may compriseone or more ZFN encoding sequences. Thus, when one or more pairs of ZFNsare introduced into the cell, the ZFNs may be carried on the same vectoror on different vectors. When multiple vectors are used, each vector maycomprise a sequence encoding one or multiple ZFNs.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered ZFPs in cells (e.g.,mammalian cells) and target tissues. Such methods can also be used toadminister nucleic acids encoding ZFPs to cells in vitro. In certainembodiments, nucleic acids encoding ZFPs are administered for in vivo orex vivo gene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome or poloxamer. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner,TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology Doerfler and Böhm (eds.) (1995); and Yuet al., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids encoding engineered ZFPsinclude electroporation, lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acidconjugates, naked DNA, artificial virions, and agent-enhanced uptake ofDNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) canalso be used for delivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.) and BTX Molecular Delivery Systems (Holliston, Mass.)and Copernicus Therapeutics Inc., (see for example U.S. Pat. No.6,008,336).

Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No.4,946,787; and U.S. Pat. No. 4,897,355) and lipofection reagents aresold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Feigner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression of a ZFP fusion protein ispreferred, adenoviral based systems can be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand high levels of expression have been obtained. This vector can beproduced in large quantities in a relatively simple system.Adeno-associated virus (“AAV”) vectors are also used to transduce cellswith target nucleic acids, e.g., in the in vitro production of nucleicacids and peptides, and for in vivo and ex vivo gene therapy procedures(see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No.4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994);Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinantAAV vectors are described in a number of publications, including U.S.Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260(1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat& Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 by invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1 a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFPnucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1(granulocytes), and Iad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP nucleic acids can also be administered directly to anorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed; for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

As noted above, the disclosed methods and compositions can be used inany type of cell including, but not limited to, prokaryotic cells,fungal cells, Archaeal cells, plant cells, insect cells, animal cells,vertebrate cells, mammalian cells and human cells. Suitable cell linesfor protein expression are known to those of skill in the art andinclude, but are not limited to COS, CHO (e.g., CHO—S, CHO-K1, CHO-DG44,CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO,SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6,insect cells such as Spodoptera fugiperda (Sf), and fungal cells such asSaccharomyces, Pichia and Schizosaccharomyces. Progeny, variants andderivatives of these cell lines can also be used.

Applications

The disclosed methods and compositions can be used for inactivation of aGS genomic sequence. As noted above, inactivation includes partial orcomplete repression of GS gene expression in a cell. Inactivation of aGS gene can be achieved, for example, by a single cleavage event, bycleavage followed by non-homologous end joining, by cleavage at twosites followed by joining so as to delete the sequence between the twocleavage sites, by targeted recombination of a missense or nonsensecodon into the coding region, by targeted recombination of an irrelevantsequence (i.e., a “stuffer” sequence) into the gene or its regulatoryregion, so as to disrupt the gene or regulatory region, or by targetingrecombination of a splice acceptor sequence into an intron to causemis-splicing of the transcript.

Thus, the methods and compositions described herein allow for thegeneration of GS-deficient cell lines for use in recombinant proteinproduction, for example αl-antitrypsin and/or monoclonal antibodyproduction. Additional genes, such as FUT8 may also be inactivated ascells in which FUT8 is inactivated produce antibodies that exhibitgreater effector function, particularly in the induction of ADCC.

EXAMPLES Example 1 Design and Construction of ZFNs A. GlutamineSynthetase (GS) ZFNs

As the full sequence of the CHO genome is not available, the CHO GS genewas cloned and sequenced to generate the target DNA sequences for ZFNdesigns. The complete CHO GS sequence is shown in FIG. 13, which alsodesignates introns and exons.

ZFN target sites in exon 6 of the CHO GS gene were selected because thisregion encodes amino acids critical for the catalytic function of the GSprotein based on available crystal structures (see, e.g., Almassy et al.(1986) Nature 323:304-309; Gill et al. (2002) Biochem. 41:9863-9872;Liaw et al. (1994) Biochem. 33:675-681) and, as such, ZFN-mediatedmutation of this site was expected to result in loss of functional GSactivity. Partial sequence for CHO GS exon 6 is shown below. Capitalletters indicate exon sequence; small letters indicate intron sequence;and ZFN target sites 9372/9075 (Table 1, FIG. 1A and FIG. 4A) areunderlined:

(SEQ ID NO: 30) 5′-atggcactattctgttccttttcctcccctctgaagacttggcacatggggactttggttaacaagggtgatgacttaaaagtggttcagggtagaggtaagtagaacaagctaggagcttgagttggcctgaacagttagttggccttattctaaaggtcaacatgttctttctagTGGGAATTCCAAATAGGACCCTGTGAAGGAATCCGCATGGGAGATCATCTCTGGGTGGCCCGTTTCATCTTGCATCGAGTATGTGAAGACTTTGGGGTAATAGCAACCTTTGACCCCAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTTAGCACCAAGGCCATGCGGGAGGAGAATGGTCTGAAgtaagtagcttcctctggagccatctttattctcatggggtggaagggctttgtgttagggttgggaaagttggacttctcacaaactacatgccatgctcttcgtgtttgtcataagcctatcgttttgtacccgttggagaagtgacagtactctaggaatagaattacagctgtgatatgggaaagttgtcacgtaggttcaagcatttaaaggtctttagtaagaactaaatacacatacaagcaagtgggtgacttaattcttactgatgggaagaggccagtgatgggggtcttcccatccaaaagataattggtattacatgttgaggactggtctgaagcacttgagacataggtcacaaggcagacacagcctgcatcaagtatttattggtttcttatggaactcatgcctgctcctgcccttgaaggacag-3′

Binding sites for ZFN9372 and ZFN9075 are shown in FIG. 4B, while FIG.4C depicts the target and finger designs of these ZFNs.

In addition, ZFNs targeted to sites in exon 2 were also designed.Partial sequence for CHO exon 2 is shown below. Capital letters indicateexon sequence; small letters indicate intron sequence; and ZFN targetsites 8365/8361 (Table 1, and FIG. 1A) are underlined.

(SEQ ID NO: 31) 5′-ggctggcagatctccgagttcgaggctgacctggtctgaatagcaaggaaattaaggggtgaggcgtatgtctgttaaagcaagaataaaaggcaaaggaacactccacagtcaattattcaagtcttgatggcagtaatgtagttgtattgggtggattaagacattctaataatgaatttttttgtctattgttccctcttttcagctttctcaaaattaatggatattaaaaatccccttagccgggcgttggtggcacacacctttaatcccagcactcgggaggcagaggcaggcagatctctgtgagttcgaggccagcctggtctccagattgagtgccaggataggctccaaagctacacagaaaccgtgtctcgaaaaacaaaacaaaaaaataaaaaaaaaaatcccttaactagcccaacctacaagggatgatctttgtctaactatgaactttaaacctcttgaaagcagagtgaataatgcacttcaataatgttgacttccaaaggagagaccaccacaccgttccctgtgcctcttacgcaattcctgcaggggacccccttcagagtagatgttaatgaaatgacttttgtctctcCAGAGCACCTTCCACCATGGCCACCTCAGCAAGTTCCCACTTGAACAAAAACATCAAGCAAATGTACTTGTGCCTGCCCCAGGGTGAGAAAGTCCAAGCCATGTATATCTGGGTTGATGGTACTGGAGAAGGACTGCGCTGCAAAACCCGCACCCTGGACTGTGAGCCCAAGTGTGTAGAAGgtgagcatgggcaggagcaggacatgtgcctggaagtgggcaagcagcctgagatttgaccttccttctgttttgtttgcaaagtctttcaaaagcaggtctcttcaggcctcagtcagtcacccgtaagctgccgagtagtctggaggcatagaaaacaatggaggcctttatttagatggaatcttgtgtgtgctggtacactgaagaaaaatattgggtcatatttgtagggggtgggaggttggagtattgctaacctagccaaccccaggaacctagtttgaaagacctgtaactagaatatgctatcaagtttatagagcagtggttctcaacctttcaaatgattacacttgaatacaactcctcatgttctggtgattacccccatcccaaccattgctaacttettaactgaaatttcactactgctacgaatcataatgtatctgtgtttttggatggtcttaggtgacccctgtgaaagggttgtgagaccatcctcaaaggggttgtgacctacaggttgagacccttttgagtgctgtgtttattagtatttatacagtggaattctgggtgcaaagcacatgctccaaagtagtttctctgggactggccatttgttttcgatggggatcttttaaaacttgcaaaggaaccaaaaaaaaaaaaatgcagaaaaaaggaggtgggggagtgcacgcctttaatcccagtacttgggaggcagaggcaggcggatctctgtgagtttgagaccagcctggtctacaagagctagttccaggacagcctccaaagccacagagaaaccctgtctcaaaacaaacaaacaaacaaaaaaattaaaaaaaaaaaaaactttcaaaggagacctgttttattttagttgtggcctttgttttggtaggaagggcagctagtttaggatgagtttttattattc taagatgttgccgtttg-3′

ZFNs targeting the CHO GS sequence (for example as shown in Table 1),were assembled into mammalian expression vectors as described in U.S.patent application Ser. No. 12/218,035 and tested by transienttransfection into CHO cells.

B. DHFR ZFNs

ZFNs targeted to DHFR were designed and produced as described in U.S.Patent Publication No2008/0015164. For experiments described herein,DHFR ZFN pairs designated 9461/7844 and 9476/9477 (individual ZFNs shownin Table 2) were used.

TABLE 2 Exemplary zinc finger nucleases targeted to DHFR ZFN nameTarget sequence F1 F2 F3 F4 ZFN 7844 QSGALAR RSDNLRE  QSSDLSR TSSNRKTccAATGCTCAGGTAct (SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID NO: 32)NO: 14) NO: 3) NO: 2) NO: 25) ZFN 9461 RSDTLSE NNRDRTK  RSDHLSA QSGHLSRagGGAAGGTCTCCGtt (SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID NO: 33)NO: 34) NO: 35) NO: 36) NO: 37) ZFN 9476 QSSDLSR DRSDLSR  QSGALARRSDHLTT caTGGGTAGCCGCTga (SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID (SEQ ID NO: 38) NO: 2) NO: 39) NO: 14) NO: 40) ZFN 9477 RSDHLST RSAHLSR RSDHLSR DSSDRKK agTCCGGGGGGTGGtg (SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID (SEQ ID NO: 41) NO: 7) NO: 17) NO: 42) NO: 43)

C. FUT8 ZFNs

ZFNs targeted to FUT8 were designed and produced as described in U.S.Ser. No. 12/218,035. For experiments described herein, FUT8 ZFNsdesignated 12176 and 12172 and shown in Table 3 were used.

TABLE 3 Exemplary zinc finger nucleases targeted to FUT8 ZFN nameTarget sequence F1 F2 F3 F4 F5 F6 ZFN 12172 RSDNLSV QNATRIN RSDHLSQQSATRTK   RSDNLSR RNDNRKT AAGGAGGCAAAGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID CAAAG NO: 45) NO: 46) NO: 9) NO: 47) NO: 48) NO: 49)(SEQ ID NO: 44) ZFN 12176 RSDNLRE NNTQLIE TSSILSR RSDNLSA RKDTRIT N/AAAGAAGGGTCATC (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID AG NO: 3) NO: 51)NO: 52) NO: 53) NO: 54) (SEQ ID NO: 50) ZFN 12170 RSDNLSV QNATRINRSDNLST QSATRTK RSDNLSR RNDNRKT taAAGGAGGCAAAG (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID ACAAAGt NO: 45) NO: 46) NO: 57) NO: 47) NO: 48) NO: 49) (SEQ ID NO: 56)

Plasmids comprising sequences encoding the above ZFNs were constructedessentially as described in Umov et al. (2005) Nature 435(7042):646-651via fusion to wild-type Fokl cleavage domains or obligate heterodimerFokl cleavage domains described in U.S. Patent Publication No.2008/0131962 and Miller et al. (2007) Nature Biotech. 25:778-785.

Example 2 GS-ZFN Modification of Endogenous GS

To determine whether GS-targeted ZFNs modified the endogenous GS locusas expected, CEL-1 mismatch assays were performed essentially as per themanufacturer's instructions (Trangenomic SURVEYOR™). Briefly, theappropriate ZFN plasmid pairs were transfected into CHO K−1 cellsgrowing adherently in serum-containing medium or CHO—S cells growing insuspension in serum-free chemically defined medium.

CHO K−1 cells were obtained from the American Type Culture Collectionand grown as recommended in F-12 medium (Invitrogen) supplemented with10% qualified fetal calf serum (FCS, Hyclone). CHO—S cells werepurchased from Invitrogen (Carlsbad, Calif.) and were grown andmaintained as suspension cultures in chemically-defined protein-free andanimal component-free CD-CHO medium supplemented with 8 mM L-glutamineand HT supplement as necessary (100 μM sodium hypoxanthine and 16 μMthymidine) (all from Invitrogen, Carlsbad, Calif.) in ahumidity-controlled shaker incubator (ATR, inc., Laurel, Md.) at 125 rpmwith 5% CO₂ at 37° C.

Adherent cells were disassociated from plasticware using TrypLE Select™protease (Invitrogen). For transfection, one million CHO K−1 cells weremixed with 1 μg each zinc finger nuclease and 100μL Amaxa Solution T.Cells were transfected in an Amaxa Nucleofector II™ using program U-23and recovered into 1.4 mL warm F-12 medium+10% FCS.

Genomic DNA was extracted from the ZFN-treated cells using the QiagenDNeasy™ kit (Qiagen, Inc.), the target locus amplified by PCR using theappropriate primers for the region of the GS locus targeted by the ZFNs,the PCR products were subjected to a melting/annealing step resulting inthe formation of distorted duplex DNA through random re-annealing ofmutant and wild-type DNA. CEL-I enzyme (Surveyor™ mutation detectionkit, Transgenomic, Inc.) was then added to specifically cleave the DNAduplexes at the sites of mismatches. The CEL-I cleaved samples wereresolved on a 10% TBE polyacrylamide gel, stained with ethidium bromide,and the DNA bands were quantified using densitometry. The frequency ofNHEJ was calculated as described essentially in Miller et al. (2007),supra.

As shown in FIGS. 1A and 1B, GS ZFNs modified the endogenous GS locus.In particular, the ZFN pair including ZFN 9372 and ZFN 9075 resulted inmodification of 26% (with wild-type cleavage domains) and 24% (withengineered obligate heterodimer forming cleavage domains) of chromosomesin CHO-K¹ cells and 25% of chromosomes in CHO—S cells (FIG. 1B).Similarly, ZFN pair including ZFN 8361 and 8365 (targeted to exon 2)resulted in modification of 7% of chromosomes.

In agreement with the results of the Surveyor™ Nuclease Assay (FIG. 1A),direct sequencing confirmed 34% (91/266) of the alleles harboredmutations at the ZFN target site typical of NHEJ-mediated DNA repair(see, e.g., Weterings et al. (2004) DNA repair (Amst) 3:1425-1435).Importantly, 81% of the sequenced mutations resulted in a reading frameshift (FIG. 5).

Example 3 Generation of GS-negative cell lines

To generate CHO cell lines lacking GS, single-cell derived lines wereisolated by limiting dilution from both the CHO-K1 and CHO—S transfectedpools described in Example 2. Sequencing of the ZFN target regionrevealed that 17 of 54 CHO—S derived cell lines (31%) had at least onedisrupted GS gene, 8 of 54 (15%) were homozygous for a mutant allele, 2were compound heterozygous, and the remaining 7 were heterozygous (withone wild-type allele). For CHO-K1-derived cell lines, sequencinganalysis identified 18 of 50 (36%) had at least one disrupted GS allele,with 5 (10%) homozygous for a given mutation. See, also, FIG. 1C showingthe genotypes of the homozygous mutant lines.

All homozygous mutant lines harbor GS mutations that result in a shiftin the open reading frame or an in-frame deletion of critical aminoacids (e.g. lines B4, KA2, and KA4), and were therefore expected toproduce no active GS enzyme. As predicted, none of the homozygous mutantcell lines grew in the absence of exogenous L-glutamine, in contrast towild-type CHO cells (FIG. 1C).

Western blot analysis using an anti-GS monoclonal antibody (BDBiosciences) confirmed that all cell lines with frame shifts or largedeletions expressed no detectable GS protein (for example, clone B3,FIG. 1D). Lines harboring small in-frame deletions (B4 and KA2) didexpress GS proteins that were detectable by Western blot but, consistentwith the elimination of amino acids critical for GS catalytic activity,did not support the growth of the cells in the absence of L-glutamine(FIG. 1C). Growth of cell line B3 was also shown to be dependent onglutamine supplementation (see FIG. 6).

To further validate their GS^(−/−) phenotype, three of theCHO-K1-derived clones were adapted for growth in suspension in achemically-defined serum-free medium over a 3 month period. Growth andviability of the cells was then monitored intensively for a further 4weeks (FIG. 1E).

The ZFN-generated GS^(−/−) lines grew normally in serum-free suspensioncultures in the presence of L-glutamine (24-27 hour doubling timesimilar to the industry standard CHOK1SV grown under similarconditions). Subsequent removal of L-glutamine resulted in immediatecessation of cell growth and a rapid loss of viability (see arrows inFIG. 1E). Transient transfection of a human IgG antibody expressionconstruct confirmed all three GS^(−/−) lines supported comparabletransgene expression levels to those obtained with the CHOK1 SV line.

Taken together, these data confirm the successful generation ofgenetically and phenotypically validated GS knockout CHO cell lines.

Example 4 Generation of GS-DHFR double knockout cell lines

To create a double-knockout cell line, ZFNs targeted to the DHFR genewere used on the background of the CHO—S GS^(−/−) cell line B3.ZFN-mediated knockout of DHFR using the strategy described for GS aboveis described in U.S. Patent Publication No. 2008/0015164.

In this Example, two pairs of ZFNs targeting two distinct regions of theDHFR gene were delivered simultaneously with the goal of eliminating theintervening genomic fragment through the repair of the cleavedchromosomal ends via NHEJ. Deletion of a ZFN-specified intergenicsequence was expected to result in a larger and better defined mutationthat eliminates the potential for a smaller in-frame mutation to recoverprotein expression e.g. FIG. 1D (Left Panel labeled “B4”).

In addition to the previously described ZFN pair 9461/7844 that targetexon 1 of the CHO DHFR gene (U.S. Patent Publication No. 2008/0015164),a second ZFN pair was generated which targets a site ˜240 by away in theintron immediately following exon 1 (FIG. 2A and FIG. 7A).

Transient transfection was performed as described in Example 3 andtransfection with either the ZFN9461/ZFN7844 pair targeting exon 1, orthe ZFN9476/ZFN9477 (See FIGS. 7B and 7C) pair targeting the intronicsite alone resulted in allelic mutation frequencies at the endogenousDHFR locus of 15% and 18%, respectively in the GS^(−/−) cell line B3(FIG. 2B). Co-transfection of both ZFN pairs into the GS^(−/−) cell lineB3 resulted in the appearance of a shorter PCR amplification productconsistent with the expected ˜240 by deletion of the sequence betweenthe ZFN binding sites (FIG. 2C). The frequency of this deletion eventwas estimated at ˜8% of all alleles and was observed only when bothpairs of ZFNs were introduced into the cells (FIG. 2C).

Cloning and sequencing of the presumptive deletion PCR fragment revealedthe expected excision of the sequence between the ZFN target sites (FIG.8). The observed minor variations in the sequence at the junction of therejoined chromosomal ends are consistent with the expected smallinsertions and deletions characteristic of the NHEJ repair process.

Single-cell derived lines were isolated from the ZFN-treated pool inFIG. 2C Lane 7 by limiting dilution. PCR-amplification and sequencingrevealed 9% (18 of 200) of the cell lines possessed the expected ˜240-bpdeletion. Four of these cell lines (2% of all lines screened, 22% of alllines containing the deletion) were found to harbor a biallelic mutationin the DHFR gene (FIG. 2D). Cell line 2B12.8 is homozygous for a 244-bpdeletion of the intervening region between the exon1 and intron 1 ZFNcleavage sites. The remainder of the lines had just one allele with the240-bp deletion while the other presented classical smaller NHEJ drivenmutations at the exonic and/or intronic ZFN target sites. For example,cell line 1F1.6 contained a ˜240-bp deletion in one allele (consistentwith a dual ZFN cleavage and deletion event), but the other allelecontained a 4-bp deletion in exon 1 resulting in a frame shift.

Cell lines 1F1.6 and 2B12.8 had genotypes consistent with complete geneknockout and thus were selected for further characterization. Westernblot analysis revealed no full-length DHFR or GS protein in either ofthese single-cell derived lines (FIG. 2E). Furthermore, neither line wasstainable with fluorescein-labeled methotrexate which binds to theactive site of the DHFR protein, in contrast to the parental GS^(−/−)line B3 or wild-type CHO cells. Most importantly, growth of the 1F1.6and 2B12.8 cell lines was dependent upon the addition of exogenoushypoxanthine and thymidine (HT) and L-glutamine to the culture medium.In contrast, the parental GS^(−/−) line B3 required L-glutamine but notHT, while the DHFR-negative CHO cell line DG44 (GS^(WT)) required onlyHT but not L-glutamine and the wild-type CHO—S required neithersupplement for growth (FIG. 2F).

The dependence on exogenous HT and L-glutamine confirms the functionalloss of both GS and DHFR activity in cell lines 1F1.6 and 2B12.8.

Thus, taken together these data demonstrate the successful generation ofgenetically and phenotypically validated GS^(−/−)/DHFR^(−/−) doubleknockout CHO cell lines.

Example 5 Generation of GS-DHFR-FUT8 triple knockout cell lines

A triple knockout of GS, DHFR and FUT8 was also generated using theGS^(−/−) DHFR^(−/−)CHO cell line IF1.6. As described in U.S. Ser. No.12/218,035, aberrant glycosylation resulting from the use of non-humancells e.g. CHO in the expression of protein therapeutics can alter thepotency, half-life and even the immunogenicity of the protein product.Thus methods for humanizing the glycosylation of proteins in non-humanexpression systems are highly desirable. The CHO FUT8 gene is such atarget encoding an α1,6-fucosyltransferase that catalyzes the transferof fucose from GDP-fucose to the core GlcNAc of N-linkedoligosaccharides. Point mutations of amino acid residues in the highlyconserved Fut motif II encoded in exon 10 result in the inactivation ofthe FUT8 enzyme.

Thus, we cloned and sequenced the region spanning FUT8 exon 10 andflanking introns from CHO-K1 genome, and designed ZFNs to target exon 10(U.S. patent application Ser. No. 12/218,035; FIG. 3A and FIG. 9).Transient delivery of ZFN12172/ZFN12176 into cell line 1F1.6 resulted inmodification of 7.5% of the FUT8 alleles (FIG. 3B), and single-cellderived lines were obtained by limiting dilution from this pool.

C. griseus FUT8 was not detectable using commercially availableantibodies, so we further assayed α1,6-fucosyltransferase activity usinga FACS-based fluorescent Lens culinaris agglutin (F-LCA) binding assay,as described in Yamane-Ohnuki et al. (2004)Biotechnol Bioeng 87:614-622.LCA selectively binds to cells that present core fucosylatedoligosaccharides on their cell surface. Cells lacking core fucosylationdue to a complete loss of FUT8 activity do not bind to LCA. Screening ofthe ZFN-generated cell lines for their ability to bind F-LCA showed noF-LCA binding in 2% of the line (4 of 200) indicating a complete absenceof FUT8 enzymatic activity (FIG. 3C).

Sequencing of PCR amplicons of genomic DNA confirmed that both clonespossess biallelic compound heterozygous mutations at the FUT8 targetlocus (FIG. 3D), which encode a shift in reading frame in thisfunctionally critical region. Thus the genotype of both cell lines isconsistent with the lack of F-LCA binding observed (FIG. 3C) anddemonstrates the ZFN-driven genetic and functional knockout of FUT8 incell lines 35F2 and 14C1. Both of the FUT8 clones also stably maintainedtheir GS^(−/−) and DHFR^(−/−) genotypes and phenotypes, throughout themultiple rounds of ZFN transfection and single-cell cloning. Indeed, thegeneration of a triple knockout cell line was well-tolerated as measuredby growth rate studies in serum-containing medium supplemented with HTand L-glutamine which gave mean population doubling times of 21.8 and21.6 hours for clones 35F2 and 14C1, respectively. Taken together thesedata demonstrate the successful generation of single, double and triplegene knockouts in CHO cells using engineered ZFNs.

Example 6 Generation of GR-CCR5—PPP1R12c triple knockout cell lines

A triple knockout cell line was also generated by concurrentadministration of inactivating ZFNs. In particular, K562 cell lines inwhich CCR5, glucocorticoid receptor (GR) and PPP1R12c (also known as theadeno-associated virus integration site or “AAVS1”) were inactivatedwere also generated by simultaneous application of ZFNs targeted tothese loci.

ZFNs targeted to CCR5, GR and AAVS1 were generated as described in U.S.Patent Publication Nos. 20080159996 (CCR5); 20080188000 (GR) and U.S.application Ser. No. 12/150,103 (PPP1R12c/AAVS1). Schematics showing theZFN binding sites in portions of these genes are shown in FIG. 10A. TheCCR5 gene contains 3 exons, the coding sequence (CDS) is located withinexon 3, and the CCR5ZFN target sequence is located in the CDS asindicated. The GR gene contains 9 exons, and the GR ZFN target sequenceis located in Exon3. The AAVS1 ZFN target sequence is located in themiddle of the AAVS1 region.

Plasmids encoding pairs of ZFNs linked to either the wild-type, ZFN-FokI(wt) or an obligate heterodimer EL/KK variant, ZFN-Fok I(EL/KK) of thecatalytic domain of Fok I were transiently co-transfected into K562cells, and the modification frequency at CCR5 (left), GR (middle), AAVS1(right) loci was determined by the Surveyor™ Nuclease Assay 10 daysafter transfection, respectively. As shown in FIG. 10B, the target geneswere subject to NHEJ following ZFN treatment.

Single-cell derived cell lines of these triple-knockout cells were alsoevaluated by PCR and sequencing of genomic DNA described above. Inparticular, the lane 8 samples in FIG. 10B, treated with CCR5ZFN-FokI(EL/KK)+GR ZFN-Fok I(EL/KK)+AAVS1 ZFN-Fok I(EL/KK) were examined. ThePCR primers were designed to specifically amplify unmodified wild-type(wt) sequences but not sequences with deletions.

Results are shown below in the following Table 4. All clones containingwt sequences (wt or heterozygous) had visible PCR bands at predictedsizes, whereas knock-out (KO) clones had no visible PCR bands. Among 144single cell clones screened by CCR5PCR, 5 clones contain CCR5KO. Nineclones were identified as GR KO by GR PCR. Two of the clones containboth CCR5KO and GR KO. All 12 clones, which are KO clones based oneither CCR5PCR or GR PCR, were screened by AAVS1 PCR. Among the 144clones screened, one CCR5 single KO clone was identified, seven GRsingle KO clones were identified, whereas no attempt was tried toidentify AAVS1 single KO clones. One CCR5/GR dual KO clone wasidentified. Two CCR5/AAVS1 dual KO clones were identified. No GR/AAVS1dual KO clone was identified among the 144 clones, though it is verylikely that a GR/AAVS1 dual KO clone might have been identified if moreclones were screened. One CCR5/GR/AAVS1 triple KO clone (B17) wasidentified.

TABLE 4 Evaluation of single cell clones of putative triple knockoutcells CCR5 GR AAVS1 Total Clones screened 144 144 12* Total KO clones 59 3 Single KO clone 1 7 N/A CCR5/GR dual KO clone 1 1 N/A CCR5/AAVS1dual KO 2 N/A 2 clone GR/AAVS1 dual KO clone N/A 0 0 CCR5/GR/AAVS1triple 1 1 1 KO clone *12 clones that were identified as CCR5 or GR KOclones

Exemplary sequencing data of cells from triple knockout clone B17 isshown in FIG. 10C. The ZFN target sequences are underlined. ‘−’ indicatedeletions, bold letters indicate insertions.

Example 7 GS-targeted ZFNs are active in multiple species and cell types

GS-specific ZFNs as described herein (designed to human CHO cell GSsequence) were also evaluated in mouse cells. As shown in FIGS. 11A and11B, ZFN target sites in GS were well conserved across species.

ZFN pairs 8361/8365 (targeted to exon 2) and 9075/9372 (targeted to exon6) were evaluated for their ability to cleave GS in human K562 and mouseNeuro2a cells as described in Example 2 above.

As shown in FIG. 12, ZFNs designed to CHO GS sequences were functionalin other human cell types and in other species.

GS-specific ZFNs (ZFN 9075 and ZFN 9372) were also tested in humanembryonic kidney HEK293 cells as describe in Example 2. As shown in FIG.14A, the CEL-I enzyme (Surveyor™ mutation detection kit, Transgenomic,Inc.) indicated an NHEJ occurance rate of 21%.

GS knockout cell lines were made in a HEK293 cell background using thesame approach as in CHO cells using the same GS ZFNs (see Example 3).Two knockout clones, clone g17 and g52, were further characterized.Clone g52 is homozygous for an 11 bp-deletion at the GS locus. Clone 17has a 169 bp-deletion at one GS allele, and a 4 bp-insertion at theother allele. As shown in FIG. 14B, neither clone expresses GS proteinas determined by western blotting. Importantly, both clones requiredexogenously supplemented L-glutamine for growth (see FIG. 14C).

Thus, ZFNs can be employed to rapidly knock out multiple genes in asingle mammalian cell line by sequential or simultaneous inactivation ofthe target genes. The results presented here are not dependent on thecell line or the choice of ZFNs. The frequency of ZFN-driven biallelicknockout events at each individual gene (>1%), combined with the lack ofdependence on selection markers results in the ability to rapidly“stack” such genetic lesions and generate complex multi-gene knockoutcell lines previously considered impractical. The high efficiency ofgene knock out following only transient ZFN expression and the abilityto stack multiple traits in eukaryotic cells makes it feasible toperform custom genetic engineering of essentially any cell type.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

1. A method of inactivating an endogenous cellular glutamine synthetase(GS) gene in a cell, the method comprising: introducing, into a cell, ahoming endonuclease having a target site in the GS gene, wherein thehoming endonuclease binds to the target site and cleaves the GS gene. 2.The method of claim 1, wherein the homing endonuclease is selected fromthe group consisting of I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV,I-CsmI, I-Pan I, I-SceII, I-PpoI, I-SceIII, 1-CreI, I-TevI, I-TevII andI-TevIII.
 3. The method of claim 2, wherein the homing endonuclease isengineered to bind to the target site.
 4. The method of claim 1, whereinthe homing endonuclease is introduced into the cell as a polynucleotide.5. The method of claim 1, further comprising inactivating a DHFR gene inthe cell using a nuclease.
 6. The method of claim 1, further comprisinginactivating a FUT8 gene in the cell using a nuclease.
 7. The method ofclaim 5, further comprising inactivating a FUT8 gene in the cell.
 8. Amethod of producing a recombinant protein of interest in a host cell,the method comprising the steps of: (a) providing a host cell comprisingan endogenous GS gene; (b) inactivating the endogenous GS gene of thehost cell by the method of claim 1; and (c) introducing an expressionvector comprising a transgene, the transgene comprising a sequenceencoding a protein of interest into the host cell, thereby producing therecombinant protein.
 9. The method of claim 8, wherein the protein ofinterest comprises α1-antitrypsin.
 10. The method of claim 8, whereinthe protein of interest is a monoclonal antibody.
 11. A cell line inwhich a GS gene is partially or fully inactivated, wherein the cell lineis produced by (a) inactivating the GS gene in a cell according to themethod of claim 1; and (b) culturing the cell under conditions suitablefor generating a cell line in which the GS gene is partially or fullyinactivated.
 12. The cell line of claim 11, wherein the cell is amammalian cell selected from the group consisting of a COS cell, a CHOcell, a VERO cell, a MDCK cell, a W138 cell, a V79 cell, a B14AF28-G3cell, a BHK cell, a HaK cell, a NSO cell, a SP2/0-Ag14 cell, a HeLacell, an HEK293 cell, and a perC6 cell.
 13. The cell line of claim 11,further comprising a nuclease-inactivated FUT8 gene.
 14. The cell lineof claim 11, further comprising a nuclease-inactivated DHFR gene. 15.The cell line of claim 11, further comprising nuclease inactivated FUT8and DHFR genes.